Gel-forming polypeptide derivatives

ABSTRACT

The invention relates to N-terminal Fmoc-protected peptide combinations that form gels in water and diverse organic solvents, and whose representative overall formulas are: (I), where R 1  is CH 3 , CH 2  --CH(CH 3 ) 3 , CH(CH 3 )CH 2  CH 3 , R 2  is H, CH 3 , CH 2  OH, (CH 2 ) n  --COOH, (CH 2 )4--NH--CO--OCH 2  C 6  H 5 , R 3  is dipeptide remainder, m is 0 or 1 and, n is 1 or 2; or (II), where R1 is CH 3 , CH 2  --CH(CH 3 )2, or CH(CH 3 )CH 2  CH3, R2 is CH2--CH(CH3)2, R3 is H, CH 3 , CH 2  OH, (CH 2 ) n  --COOH, or (CH 2 )4--NH--CO--OCH 2  C 6  H 5 , R 4  is tripeptide remainder, m is 0 or 1 and, n is 1 or 2. These types of peptides form gels in aqueous solutions and are biologically compatible and may be useful for drug delivery, antigen delivery and may be useful as food additives to retard spoilage and act as fillers.

TECHNICAL FIELD

The invention comprises a series of chemically synthesized low moleculeweight peptides that form gels in water or organic solvents, as well asthe gels so formed. The molecular structure of these gels can beexploited for new materials, stereocatalytic matrices and micellepreparation for research, medical, cosmetic, and food productapplications.

BACKGROUND OF THE INVENTION

Gels formed by biocompatible materials have many applications inmedicine and industry, but have been limited by the need for highmolecular weight compounds, high concentrations of polymer, or organicsolvents to form the gels. It would be desirable to synthesize gels thatform in aqueous solutions at low concentrations of simple polymers.

A gel consists of continuous networks of molecular aggregates in whichsolvent molecules are trapped. The gel phase requires stereochemicalcorrespondence between these small molecules in order to generatecohesion. Low molecular weight molecules that form macroscopic gels maydo so by a variety of microscopic packings including linear aggregates,micelles, and other structures. Components responsible for themechanical elasticity of gels may be joined by fragile non-covalentbonds, yielding materials with mechanical properties suitable for pastesor spreads with many cosmetic or therapeutic applications.

High molecular weight network systems are also well described which formgels in water, such as gelatin and starch. These gels form when themacromolecules are mixed with water, heated, and cooled.

There are several examples of low molecular weight compounds that formgels, but predominantly in toxic organic solvents. The chemicalstructures of such compounds are varied, including for example, organicacids with long aliphatic chains, partially fluorinated n-alkanes,cholesterol derivatives, oxyanthracene derivatives, and others asdiscussed by Hanabusa, K., et al., J. Chem. Soc. Chem. Commun. 4,390-392 (1993).

Among the low molecular weight compounds that form gels are amino acidsand peptide derivatives. Already described in patents to Saito T., etal., Jp. 50022801 (1975) and Saito T., et al., U.S. Pat. No. 3,969,087(1976) are the structures of amino acid esters and amides containingfatty acids and higher alcohols which form gels in oils and otherorganic solvents.

Hanabusa K., et al., J. Chem. Soc., Chem. Commun. (18)1371-1373 (1992)discloses that the alanine derivative N-benzyl-oxycarbonyl L-alanine4-hexadecanoyl-2nitrophenyl ester forms thermally reversible gels inmethanol and cyclohexane at concentrations below 1%. Similarly,Hanabusa, K., et al., J. Chem. Soc., Chem. Commun. 4, 390-392 (1993)discloses that N-benzyloxycarbonyl L-valyl-valine n-octadecylamide makesgels in several organic liquids.

Depsipeptides with the formulas (X--X¹ --OCH₂ CH₂ COO)_(n) where X andX¹ can be valine or isoleucine derivatives form thermostable gels withsuch solvents as methylene chloride, acetonitrile, ethylacetate, andacetone as discussed in De Vries E. J., et al., J. Chem. Soc., Chem.Commun. (3) 238-240 (1993).

Ihara H., et al., J. Chem. Soc., Chem. Commun. (17) 1244-1245 (1992)relates to benzyl-oxycarbonyl β-alanine glumamate derivatives in whichboth carboxyl groups are derivitized with N-dodecylamide groups,dissolve in hot benzene, but on cooling these mixtures form gels.Notably less common are low molecular weight compounds that form gels inwater. Among these is the synthetic glycolipid N-octyl-D-gluconamide,which upon prolonged heating in water solutions partly hydrolyses.

It is also noted that t-butoxycarbonyl-valyl-valylisoleucine methylesterforms micelles in chloroform in Jayakumar A., et al., J. Chem Soc.,Chem. Commun. (10) 853-855 (1993). The critical micelle concentration ofthis peptide is 2.5 mM.

Kalopissis G., et al., French Patent 1,397,231 (1965) disclosesasparagine derivatives whose amino groups are acylated by fatty acidsand whose amide groups are alkylated. These derivatives form gels inwater at concentrations around 3%.

Mandal, A. B. and Jayakumar, J., J. Chem. Soc., Chem. Commun. 3, 237-238(1993) has shown that the tetrapeptide Tyr-Gly-Phe-Ala benzylesterdiluted in trifluoroacetate/water mixtures forms micelles. WhileWeitzberg M., et al., PCT Int. Appl. 90 15,602 (1990) and Burch R. M.,et al., Proc Natl. Acad. Sci., USA, 88 (2) 355-359 (1991) show that some9-fluorenylmethoxycarbonyl-(Fmoc-) amino acid derivatives modulate theimmune response, Noronha-Blob L., et al., Eur. J. Pharmacol., 199(3)387-388 (1991) has also shown these derivatives have potential to reduceendotoxic shock.

Notably absent from current gel-forming systems is the ability torapidly and inexpensively form gels from low molecular weight compoundsat low concentrations that are stable in aqueous, non-toxic solutions.Such gels would have many applications in medicine and industry.

SUMMARY OF THE INVENTION

It is the object of the present invention to provide an inexpensive andreadily produced biologically compatible gelling substance. This isaccomplished by utilizing the newly developed N-terminal Fmoc-protecteddipeptide or tripeptide compound series presented here. When placed inan aqueous solution individual dipeptide or tripeptide moleculesaggregate in a manner that creates rodlike micelles that can encapsulateother solutes. Similarly shaped micelles have been observed in solutionsof larger peptide derivatives, but only in organic solvents as discussedin Mandal A. B., et al, J. Chem. Soc., Chem. Commun. (3) 237-238 (1993)and Hanabusa, K., et al., J. Chem. Soc., Chem. Commun. 4, 390-392(1993).

Another object of this invention is to provide a novel delivery systemfor small hydrophobic or amphiphilic therapeutic molecules. These drugsmay range in molecluar weight from approximately 100 Da to approximately5000 Da. The invention is compatible with either topical or circulatorytherapeutics. Many drugs are currently delivered using liposome-basedvectors, which are both more expensive to produce and more difficult tocreate.

A further object of the present invention is to provide a vehicle forinjected antigens that boosts their immuno-stimulatory abilities in theabsence of conventional crosslinking and adjuvent.

The stable aqueous gels using the dipeptide or tripeptide compoundsdescribed herein can also be used, for example, as food additives toretard spoilage by slowing water loss, as food thickening agents, in thepurification of proteins by ion exchange chromatography, in the affinitypurification of antibodies from serum, and as a malleable cream base forcosmetics.

SUMMARY OF THE FIGURES

FIG. 1 shows the characteristic oscillatory motion of a solution of thepartly protected dipeptide Fmoc-L-Leu-L-Asp (compound 5 of Table 2)obtained from a rheologic measurement using a torsion pendulum. Fromthis curve is calculated the shear modulus G¹.

FIG. 2 shows the dependence of G¹ on peptide concentration.

FIG. 4 shows dependence of light scattering intensity on temperature fora compound 5 gel. Experimental parameters: 633 nonometer light,scattering angle--90°, Compound 5 concentration=0.37 mg/ml in 10 mMtris-buffer at pH 7.4.

FIG. 5 shows the effects of compound 5 and gelatin on the evaporation ofwater.

FIG. 6A shows the light scattering intensity of various concentrationsof Fmoc-Leu-Asp in 10 mM Tris pH 7.0, measured at 20° C. (circles) and60° C. (triangles).

FIG. 6B shows the storage (closed symbols) and loss shear moduli (opensymbols) of 2 mg/ml Fmoc-Leu-Asp in 10 mM Tris pH 7.0 at 60° C.

FIG. 6C shows the measurement of shear storage moduls, G' (closedsymbols) and shear loss modulus, G" (open symbols) at 1% maximal strainover a range of frequencies at 60° C. (triangles.) and 20° C. (circles).Other experimental conditions are as described for FIG. 6B.

FIG. 6D shows the measurement of G' and G" at various maximal shearstrains at a frequency of 1 rad/s. Other experimental conditions andsymbols are as described for FIG. 6C.

FIG. 7. Confocal scanning micrograph of 4 mg/ml Fmoc-Leu-Asp in 10 mMTris pH 7.0 containing also 0.02 weight fraction rhodamine-Leu-Asp.

FIGS. 8A and B show the development of antibodies against5-methyl-1adamantanamine 3-carboxylic acid hydrochloride (AdaMeC) usingFmoc-Leu-Asp.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to N-terminal Fmoc-protected peptide combinationsthat form gels in water and diverse organic solvents, and whosedipeptide embodoiment's representative overall formula is: ##STR1##where R1 is CH₃, CH₂ --CH(CH₃)2, or CH(CH₃)CH₂ CH3

R2 is H, CH₃, CH₂ OH, (CH₂)_(n) 13 COOH, or (CH₂) 4--NH--CO--OCH₂ C₆ H₅,

R3 is dipeptide remainder,

m is 0 or 1 and,

n is 1 or 2.

This type of peptide forms gels in water at concentrations less than 1%by weight and is stable between at least 10° C. and 60° C. The gels haveelastic moduli on the order of 100 Pa, at peptide concentrations of 2-4mg/ml, and are characteristic of elastic networks formed by filamentousproteins. The concentration at which Fmoc-Leu-Asp gels form is in therange of the most efficient gelation agents known as shown in Janmey P.A. et al. Biochemistry 27, 8218-8227 (1988) and Ferry, J. D. Ann. NYAcad. Sci. 408, 1-10, (1983), implying that highly elongated structuresmust form. Thin rodlike filaments with lengths of 10 microns arevisualized by incorporation of trace amounts of rhodamine-Leu-Asp intothe Fmoc-Leu-Asp gel as shown in FIG. 7. FIG. 7 is a confocal scanningmicrograph of 4 mg/ml Fmoc-Leu-Asp in 10 mM Tris pH 7.0 containing also0.02 weight fraction rhodamine-Leu-Asp obtained with a BioRad MRC 600confocal imaging system attached to a Zeiss inverted microscope.

In contrast, gelatin solutions remain sols at such low concentrations,and the lowest concentration of other synthetic polymer gels withcomparable shear moduli are at least an order of magnitude higher.Individual examples of this class of peptides form gels in organicsolvents such as diethylether, hydrocarbons, and their mixtures.

Of particular interest in this invention are the gels formed withFmoc-Leu-Asp. At concentrations between 1 mg/ml and 1.5 mg/ml solutionsof Fmoc-Leu-Asp undergo an abrupt increase in light scattering at both20° C. and 60° C., as shown in FIG. 6A wherein 1 ml solutions in 1 cmdiameter cylindrical cells were measured with a Brookhaven InstrumentsBI30ATN apparatus using a 633 nm 10 mW laser. When a solution of 2 mg/mldipeptide is cooled from 100° C. to 60° C., viscoelastic parameterscharacteristic of a gel are obtained by low strain oscillatorymeasurements. The storage shear storage modulus, G', a measure of theelastic strength of the material, reaches a stable level of 80 Pa withinseveral minutes (FIG. 6B). These measurements were made using aRheometrics RFSII instrument1. The maximal shear strain was 1% foroscillatory deformation at a frequency of 1 rad/s. This value iscomparable to that of the strongest biopolymer gels, such as fibrin, asshown in Ferry, J. D., Ann. NY Acad. Sci. 408, 1-10, (1983) or F-actin,as shown in Janmey P. A. et al., Biochemistry 27, 8218-8227 (1988). Incontrast, gelatin solutions remain sols at this concentration, and thelowest concentration of other synthetic polymer gels with comparableshear moduli are at least an order of magnitude higher. G', shearstorage modulus, depends weakly on frequency from 0.1 to 100 rad/s, andis much larger than the shear loss modulus G" (FIG. 6C). It is alsoinsensitive to temperature, suggesting that structures formed in thedipeptide gel are thermally stable over a physiologically relevantrange. However, as FIG. 6D shows, the shear moduli depend strongly onthe magnitude of shear deformation. The gels are strain-weakening at thesmallest measurable strains (0.3%), and although G' remains>G", bothmoduli fall by a factor of 100 when the samples are strained to 10%. Inthis sense, Fmoc-LeuAsp gels differ from fibrin, as shown in Bale, M. D.& Ferry, J. D., Thromb. Res., 52, 565-72 (1988) or F-actin, as shown inJanmey P. A. et al., Biochemistry 27, 8218-8227 (1988) gels, as thelatter both show strain hardening at 10% followed by rupture andweakening at larger strains. The structural specificity of thegel-forming peptide is summarized in Table 1.

                  TABLE 1    ______________________________________    Fmoc-dipeptides that form aqueous gels                 Melting  Optical      Gel formed                 point,   rotation,    (minimum    Sequence     ° C.                          c = 1; ethanol                                       conc., %)    ______________________________________    Fmoc--Leu--Asp                 158-160  -8.4         Yes (0.5)    Fmoc--Ala--Asp                 135-137  -5.2         Yes (6.7)    Fmoc--Ile--Asp                 162-168  -11.6        Yes (0.4)    Fmoc--Leu--Ala                 168-170  -26.5        No    Fmoc--Leu--Bal                 150-152  -25.3        No    Fmoc--Leu--Glu                 100-102  -16.9        No    Fmoc--Leu--Lys(Cbz)                  98-100  -12.3        No    ______________________________________

The partially protected dipeptide Fmoc-Leu-Asp and its analogs set forthin Table 1 were synthesized in 60-80% yield by reaction of thecorresponding Fmoc-amino acid-O-succinimidyl esters with sodium salts ofamino acids or their derivatives. The reaction was carried out inaqueous DMF, and the peptides were purified by HPLC in anacetonitrile/water gradient.

Further peptide variants of this invention can be synthesized by any ofseveral methods well known in peptide chemistry. One of these methods,used for compound 1 of following table 2 is the following:

An N-protected amino acid N-hydroxysuccinamide ester is formed from anN-protected amino acid and N-hydroxysuccinamide by the carbodimidemethod in demethylformamide (DMF). The ester is coupled to another aminoacid in aqueous KOH solution. After a few hours, the activated ester hasreacted and the reaction product can be separated as mentioned infollowing Example 1.

Other types of activated esters can also be used, other blockers ofamino acid carboxyl groups can be used, and other solvents can beemployed.

Aqueous gels of the synthesized compounds can be made by several methodswhich are outlined here and more fully described in the examples.

1. Finely ground dipeptide or tripeptide compound is suspended inboiling water, the mixture is briefly and vigorously mixed or shaken andleft to cool. Between 50-60° C. gelation occurrs. This procedure can berepeated to obtain a more homogeneous mixture and a stronger gel.

2. The dipeptide or tripeptide compound is dissolved in a minimal volumeof organic solvent which is miscible in water, and at room temperature asmall volume of the solution is added to a large volume of water withrapid mixing, after which the gel forms.

3. Methods 1 and 2 can be combined.

The amount of Fmoc-protected dipeptide or tripeptide required for gelformation is 0.1% to 5% by weight. The amount of compound incorporatedinto the gel usually does not exceed 1% and depends on the method ofpreparation and on other additives in the mixture.

To obtain gels with specific biological effects the gels can be formedin various aqueous suspensions, solutions, and emulsions containingother solutes such as drugs or antigens. Example 4, which follows,describes boric acid-containing gels. Similarly, salicylic acid, Ssulfur compounds, and suspensions of therapeutic substances can beincorporated into the gel. Of particular interest, it is noted thatcompound 5 oof Table 2 forms gels in solutions of 70% glycerol.

The gelatin peptides may also affect the freezing point of the aqueoussolutions.

To enhance the activity of various pharmaceuticals and other compounds,these can be incorporated in the gel both physically and chemically.Covalent attachment of immunologically active substances to the gellingpeptides may render them more active antigens and the immunostimulatorypotential of large polyanions has been exploited to produce antibodiesagainst low molecular weight molecules with little or no intrinsicantigenicity. Antibody production in rabbits against the antiviral drugadamantamine proceeds at least as efficiently when it is trapped inFmoc-Leu-Asp gels without additional adjuvant as when it is covalentlylinked to BSA and injected with adjuvant by conventional methods.Additionally, coupling very water soluble compounds can render them lesssoluble and polyvalent, thereby altering their pharmacokinetics.

The prepared aqueous gels dry more slowly than comparable amounts ofgelatin and starch (Example 5 below) and therefore may have novelapplications in cosmetic, medical and food product cream compositions.

A study of the viscoelastic properties of compound 5 gels are summarizedin FIGS. 1, 2, 4 and 6. The shear modulus was measured by freeoscillations using a torsion pendulum. The theory and description of themethod are described in Plazek D., Vrancke., Berge J., Trans. Soc.Rheol. 2, 38-47 (1958); Ferry J. Viscoelastic Properties of Polymers,Wiley New York (1980), and Janmey P., et al., J. Rheol. 27, 135-153(1993). Gels are formed by method 1 using Tris buffer pH7.4. The peptideconcentration in FIG. 1 is 2.5 mg/ml (0.25%). The shear nodulus derivedfrom these data is 3.1 Pa. This denotes the moderately high elasticstrength of this gel.

The elastic modulus depends on the peptide concentration (FIG. 2).Increasing concentration from 2.5 mg/ml to 5 mg/ml strongly increasesthe elastic modulus. 5 mg/ml is near the saturation concentration.

The dependence of light scattering intensity of compound 5 solutions onpeptide concentration and temperature are shown in FIGS. 4 and 6A.

An alternative embodiment of this invention relates to N-terminalFmoc-protected peptide combinations similar to those discussed above.However, this alternative embodiment is comprised of three peptides andwhose overall formula is: ##STR2## where R1 is CH₃, CH₂ --CH(CH₃)2, orCH(CH₃)CH₂ CH3

R2 is CH2--CH(CH3)2

R3 is H, CH₃, CH₂ OH, (CH₂)_(n) --COOH, or (CH₂)4--NH--CO--OCH₂ C₆ H₅,

R4 is tripeptide remainder,

m is 0 or 1 and,

n is 1 or 2.

Of particular interest is the Fmoc-Leu-Leu-Asp compound. This compoundgels in aqueous solutions of approximately 1% acid. A preferred acidsolution for forming gels with this peptide is an aqueous solution ofapproximately 1% acetic acid. In the absence of acid, the peptide formsaggregates and very weak gels, but the elasticity is strongly increasedupon the addition of acid. This feature could be exploited inapplications where it would be desirable for a liquid to solidify in anacidic environment. For example, a aqueous mixture of this peptide and asubstance to be delivered to the stomach may be taken orally in theliquid form and upon arrival in the acidic environment of the stomach,the mixture would solidify into a gel. It is understood that other thanthe requirement for an acidic environment, this type of peptide may beused to make gels according to the methods set forth above.

Further details of the invention are shown in the following examples,which are intended to be illustrative, but not limiting of the generalcharacteristics and uses.

EXAMPLES Example 1

Synthesis of N-protected peptides

Amino acids used, except glycine and b-alanine, are in theL-configuration.

N-protected amino acid (5 mmol) and N-hydroxysuccinimide (5 mmol) aredissolved in a minimal amount of DMF (8 ml) at room temperature or withgentle heating no greater than 45° C. At 0° C. 4 ml of N₁ N¹-dicyclohexylcarbodimide (5.5 mmol) in DMF is added. The mixture is keptat room temperature for 1 hour with periodic mixing. Precipitates areremoved by filtration and the C-terminal amino acid or peptide (10 mmol)is added from a solution of 2N KOH.

The reaction mixture is vigorously stirred for 2 hours at roomtemperature. A large volume (100-200 ml) of water is gradually added andthe solution acidified with 6N HCl to pH 2.0-3.0. Depending on theparticular preparation, a precipitate, gel or oil is formed. A 50-80%yield of the product can be isolated by one of the following methods.

The precipitate is filtered with a glass filter under mild vacuum,rinsed with water, dried under vacuum and, if necessary, reprecipitatedfrom ethylacetate/petroleum ether.

Gels are treated similarly except an appropriate sized (30 cm) glassfilter is used.

Oils are taken up in ethylacetate. The ethylacetate phase is washedseveral times with water, dried with Na₂ SO₂, and evaporated undervacuum. The remainder is crystallized or reprecipitated.

                  TABLE 2    ______________________________________    Various combinations of peptides synthesized    and their properties.                      Reactant Thin-         Opti-         Synthetic    amino    layer  Melting                                             cal         compound     compo-   Rf     Point  rota-    NO.  formula 1*   nent 2*  (TLC) 3*                                      ° C.                                             tion 4*    ______________________________________    1    Fmoc--Ala--Asp                      Asp      0.53(3)                                      128-130                                             -3.9    2    Fmoc--ile--Asp                      Asp      0.82(1)                                      162-183                                             -11.6    3    Fmoc--Leu--Ala                      Ala      0.23(2)                                      168-170                                             -26.5    4    Fmoc--Leu-b-Ala                      b-Ala    0.33(2)                                      150-152                                             -25.3    5    Fmoc--Leu--Asp                      Asp      0.80(1)                                      158-160                                             -8.4    6    Fmoc--Leu--Glu                      Glu      0.1(2) 100-102                                             -16.9    7    Fmoc--Leu--Gly.sub.3                      Gly.sub.3                               0.48(1)                                      182-184                                             -11.4    8    Fmoc--Leu--Lys--                      Lys(Cbo) 0.25(2)                                       98-100                                             -12.3         (Cbo)    ______________________________________     In Table 2, the following remarks apply:     1* Standard 3 Letter amino acid code. (Cbo) denotes --CO--O--Ch2C6H5 grou     2* Component 1 is Fmoc--Ala for compound 1; Fmoc--Ile for compound 2; and     Fmoc--Leu-- for compounds 3-8.     3* Silica gel TLC system:     (1) nbutanol-water-acetic acid 4:1:1     (2) chloroformethanol-ethylacetate-acetic acidwater 85:5:8:2:0.25     (3) system(2)isopropanol 4:1     4* in DMF: dimethylformamide or Ethanol

Example 2

Preparation of aqueous gels.

Boil 10 ml water and suspend in it 0.8% finely ground compound 5 ofTable 2. Strongly shake or stir the suspension then let stand 10-15minutes. On cooling the solution becomes turbid at 50-60° C. and at roomtemperature a gel forms. The gel can be reheated, agitated and recooled.When repeated several times, a stronger gel forms.

Compound 5 is soluble below 0.8%. At concentrations below 0.4% little orno gel forms. When such a solution is evaporated to 50% volume and thenheated and cooled, a gel forms.

Example 3 Preparation of gels in ethylether.

Suspend 150-200 mg finely ground compound 5 of table 2 in 10 mL boilingdiethylether. Shake suspension, filter and let sit 30-60 minutes at -5°C. On cooling a gel forms. The peptide concentration can be measured byweighing the gel, and evaporating the solvent in vacuum to a constantweight. In this way a solute concentration of 1-1.2% was found.

Example 4

Gel of compound 5 and boric acid.

Add 0.8% compound 5 to 3.5% boric acid in boiling water and shake. Oncooling, a gel forms. The gel can be smeared on a glass plate to athickness of 1-2 mm. When the water evaporates, a uniform filmcontaining the boric acid is formed.

Example 5

Gel drying.

Gels containing either 0.5-0.6% or 0.8% compound 5, gelatin, or starchare disolved in water at 90° C. and briefly cooled. The solutions (0.8ml) are poured into shallow plastic dishes (17 mm inner diameter). Onreaching room temperature, the dishes are weighed and left withdesicants but without vacuum to dry. Only compound 5 forms a gel oncooling. After 15 hours, the compound 5 gel has lost 57% of its water,but the gelatine and starch solutions have lost 70% and 87% of theirwater, respectively. The effects of compound 5 and gelatin are shown inFIG. 5. Gelatin is less effective than compound 5 at retarding theevaporation of water.

Example 6

Incorporation of antigenic agents into gels and stimulation of immuneresponse.

In discussion of this example, the following abbreviations are used:

Ada2Me: 3,5-dimethyl-1-adamantanamine hydrochloride,

AdaMeC: 5-methyl-1-adamantanamine 3-carboxylic acid hydrochloride,

AdaMeC-BSA: conjugate of bovine serum albumin and

AdaMeC prepared by the carbodiimide method,

AdaMeC-Bal-BSA: conjugate of bovine serum albumin and

AdaMeC-(beta-alanine) with a free amino group.

The low molecular weight drugs 3,5-dimethyl-1-adamantanaminehydrochloride (Ada2Me) and 5-methyl-1-adamantanamine 3-carboxylic acid(AdaMeC) can also be incorporated into the Fmoc-Leu-Asp gels (10mg/ml=21 mM) at concentrations of 1 mM and 33 mM, respectively. Athigher total concentrations, (>5 mM Ada2Me or >33 mM AdaMeC) theseagents inhibit gelation. When the Fmoc-Leu-Asp gel containing AdaMeC inphosphate buffered saline was injected into rabbits, without adjuvant,antibodies were raised against this drug to produce antisera with titersas high or higher than those of animals immunized with AdaMeC-BSAconjugates in equal volumes of complete Freund's adjuvant (FIG. 8).Three methods of antibody determination, double diffusion in agarosegel, reverse radial diffusion, and passive hemagglutination gavecomparable results. Injected alone, neither Ada2Me (5 mM) nor AdaMeC (33mM) produced specific antibodies. Fmoc-Leu-Asp alone produced antibodieswith titers no greater than 1:4.

Female random-bred rabbits were immunized three times, at weeklyintervals and bled on the 24th day. A full dose was used for the firsttwo immunizations, and a half of the full dose for the thirdimmunization. Full doses of the BSA conjugates were 100 μg totalprotein. Before injections, AdaMeC-BSA and AdaMeC-Bal-BSA solutions werediluted with equal volumes of complete Freund's adjuvant, butAdaMeC+dipeptide gel was diluted with an equal volume of 0.01M phosphatebuffer; pH 7.4. The volume of immunogen solutions injected each time was1 ml. Immune sera were analyzed by two immunoprecipitation assays, thedouble diffusion in agarose gel and the reverse radial immunodiffusion,as described by Oudin, J. Meth. Enzymol., 70, 166-98 (1980). As shown inFIG. 8A double diffusion in agarose was used to determine the titer ofantibodies recognizing AdaMeC-containing conjugates in sera of rabbitsimmunized with AdaMeC-coupled with or without a beta-alanine (Bal)spacer to BSA in complete Freunds adjuvant or with AdaMeC mixed withFmoc-Leu-Asp without adjuvant. The antisera were tested against each ofthe three immunogens and compared to sera of rabbits injected witheither AdaMeC or Fmoc-Leu-Asp-alone. And as shown in FIG. 8B the passivehemagglutination test, as described in Adler, F. L. & Adler, L. T.,Meth. Enzymol., 70, 455-66 (1980), was used to analyze antisera ofrabbits immunized with each of the three AdaMeC conjugates afterchallenge with the three AdaMeC conjugates, AdaMeC alone or Ada2Me. Inthe first two assays, the solid phase was 1% agarose containing 0.8%sodium chloride. The sample volumes were 25 μl. The probes wereincubated for 72 h at 37° C. In double diffusion in agarose gel, theantigens were placed in central wells and the antisera in surroundingwells in concentrations between 20 μg/ml and 1000 μg/ml which includedalso the equivalent balanced concentration. In the reverse radialimmunodiffusion assay, Ada2Me or AdaMeC was mixed with agarose inconcentrations between 20 μg/ml and 200 μg/ml, and the antisera wereplaced in wells in various concentrations. In control experiments, seraof non-immunized animals were used. In the passive hemagglutinationreaction, 0.5% suspensions of sheep red blood cells were treated withseven preparations: two BSA conjugates AdaMeC-BSA, AdaMeC-BalBSA!, BSAalone, Ada2Me+dipeptide gel, the dipeptide gel alone, and Ada2Me orAdaMeC alone. The protein concentration in the BSA preparations was 1mg/ml. In the case of the dipeptide gel preparations, 0.5 ml of the gelwas mixed with 4.5 ml of a suspension of SRBC. Ada2Me was applied in aconcentration of 200 μg/ml. The mixtures were incubated for 1 h at 20°C., the unadsorbed antigens were removed by centrifugation, SRBC werewashed. Samples of 50 μl were placed in the wells of the trays. Antiseraand purified antibodies were diluted with 0.01M phosphate-bufferedsaline, pH 7.4 starting from 1:100 with a step of 1/2. Untreated SRBC,sera and antibodies from non-immunized animals were used in controlexperiments.

We claim:
 1. An F-moc derivatized anionic dipeptide of the generalformula: ##STR3## wherein: R1 is selected from the group consisting ofCH₂ --CH(CH₃)₂ and CH(CH₃)CH₂ CH₃ ;R2 is selected from the groupconsisting of H, CH₃, CH₂ OH, (CH₂)_(n) --COOH, and (CH2)₄--NH--CO--OCH₂ C₆ H₅ ; R3 is OH; m is either 0 or 1; and n is either 1or
 2. 2. The dipeptide of claim 1 wherein R2 is CH₂ --COOH.
 3. Thedipeptide of claim 2 wherein R1 is CH₂ --CH(CH₃)₂.
 4. The dipeptide ofclaim 2 wherein R2 is CH(CH₃)CH₂ CH₃.
 5. An aqueous gel comprising waterand 0.1% to 5.0% by weight of the dipeptide of claim
 1. 6. An aqueousgel comprising water and 0.1% to 1.0% by weight of the dipeptide ofclaim
 1. 7. An aqueous gel comprising water and 0.1% to 1.0% by weightof the dipeptide of claim
 2. 8. An aqueous gel comprising water and 0.1%to 1.0% by weight of the dipeptide of claim
 3. 9. An aqueous gelcomprising water and 0.1% to 1.0% by weight of the dipeptide of claim 4.10. The aqueous gel of claim 5 further comprising an antigen.
 11. Theaqueous gel of claim 5 further comprising a low molecular weight drug.12. The aqueous gel of claim 11 wherein the low molecular weight drug isselected from the group consisting of 3,5-dimethyl-1-adamantanaminehydrochloride and 5-methyl-1-adamantanamine 3-carboxylic acid.
 13. AnF-moc derivatized anionic dipeptide of the general formula: ##STR4##wherein: R1 is selected from the group consisting of CH₃, CH₂ --CH(CH₃)₂and CH(CH₃)CH₂ CH₃ ;R2 is selected from the group consisting of CH₂ OH,(CH₂)_(n) --COOH, and (CH2)₄ --NH--CO--OCH₂ C₆ H₅ ; R3 is OH; m iseither 0 or 1; and n is either 1 or
 2. 14. The dipeptide of claim 13wherein R2 is CH₂ --COOH.
 15. The dipeptide of claim 14 wherein R1 isCH₂ --CH(CH₃)₂.
 16. The dipeptide of claim 14 wherein R1 is CH₃.
 17. Thedipeptide of claim 14 wherein R2 is CH(CH₃)CH₂ CH₃.
 18. An aqueous gelcomprising water and 0.1% to 5.0% by weight of the dipeptide of claim13.
 19. An aqueous gel comprising water and 0.1% to 1.0% by weight ofthe dipeptide of claim
 13. 20. An aqueous gel comprising water and 0.1%to 1.0% by weight of the dipeptide of claim
 14. 21. An aqueous gelcomprising water and 0.1% to 1.0% by weight of the dipeptide of claim15.
 22. An aqueous gel comprising water and 0.1% to 1.0% by weight ofthe dipeptide of claim
 15. 23. An aqueous gel comprising water and 0.1%to 1.0% by weight of the dipeptide of claim
 17. 24. The aqueous gel ofclaim 18 further comprising an antigen.
 25. The aqueous gel of claim 18further comprising a low molecular weight drug.
 26. The aqueous gel ofclaim 25 wherein the low molecular weight drug is selected from thegroup consisting of 3,5-dimethyl-1-adamantanamine hydrochloride and5-methyl-1-adamantanamine 3-carboxylic acid.
 27. A method for making anaqueous gel comprising:placing a dipeptide of claim 1 in water underconditions to form a gel and allowing the gel to form.
 28. A method formaking an aqueous gel comprising:placing a dipeptide of claim 13 inwater under conditions to form a gel and allowing the gel to form.